【摘要】 目的:制备兔抗果蝇Dnop5蛋白的抗体,并进行特性鉴定。方法:以RTPCR扩增Dnop5的全长cDNA并克隆入pET28a(+)表达载体中,表达并纯化Dnop5His融合蛋白。用纯化的Dnop5His融合蛋白免疫家兔,制备抗Dnop5的抗体,并用His亲和层析法进行纯化。采用Westernblot法和免疫组化染色法鉴定该抗体的特异性及生物学活性。结果:表达的Dnop5His蛋白以包涵体的形式存在,用His亲和层析法分离纯化后得到较纯的Dnop5His融合蛋白,以纯化的Dnop5His免疫家兔制备的兔抗Dnop5的抗体,经Westernblot分析显示:该抗体可与果蝇的胚胎、幼虫、蛹及成虫组织中表达的Dnop5特异性结合。结论:获得具有良好特异性的兔抗dnop5抗体,为进一步研究Dnop5的功能奠定了基础。更多还原

【Abstract】 AIM: To prepare the rabbit antibody against Dnop5 and identify its specificity. METHODS: Dnop5 cDNA was amplified by RT-PCR, and then was subcloned into the fusion expression vectors pET28a(+). After being expressed in E.coli BL21, the truncated Dnop5 protein was purifed and used to immunize rabbit. Purified antibody was obtained through affinity chromatography column with the expressed Dnop5. The specificity of the purified antibody was characterized by Western blot and immunohistochemical staining. RESULTS: The Dnop5 gene was successfully inserted into pET28a(+). After induction, the fusion protein was expressed in the form of inclusion body. The purified fusion protein was obtained by affinity chromatography. After immunization of rabbits, the antibody against Dnop5 was obtained. Western blot analysis and immunohistochemical staining showed that the antibody had a good specificity. CONCLUSION: The rabbit antibody against Dnop5 has been successfully prepared, which lays the foundation for further study on the Dnop5 function.更多还原